US7402412B2ExpiredUtilityA1

Mutated D-aminotransferase and method for producing optically active glutamic acid derivatives using the same

94
Assignee: AJINOMOTO KKPriority: Dec 9, 2002Filed: Jun 9, 2005Granted: Jul 22, 2008
Est. expiryDec 9, 2022(expired)· nominal 20-yr term from priority
Inventors:Masakazu SugiyamaKunihiko WatanabeTatsuki KashiwagiEi-Ichiro Suzuki
C12N 9/1096C12P 17/10C12N 1/20C12N 15/52C12N 15/09
94
PatentIndex Score
35
Cited by
15
References
38
Claims

Abstract

A D-aminotransferase can be modified so as to efficiently produce (2R, 4R)-monatin having high sweetness intensity from 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid by substituting an amino acid at least at one of positions (positions 100, 180 to 183, 243 and 244) involved in efficiently producing the (2R, 4R)-monatin in an amino acid sequence of a wild-type D-aminotransferase represented in SEQ ID NO:2.

Claims

exact text as granted — not AI-modified
1. An isolated DNA comprising a nucleotide sequence selected from the group consisting of:
 (A) the nucleotide sequence of SEQ ID NO:1, wherein said DNA encodes a protein having a D-aminotransferase activity; 
 (B) a nucleotide sequence encoding a protein having D-aminotransferase activity which hybridizes under stringent conditions with another DNA composed of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1, wherein said stringent conditions comprise 65° C., 0.1×SSC and 0.1% SDS; 
 (C) a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, wherein said protein has a D-aminotransferase activity; and 
 (D) a nucleotide sequence encoding a protein having an amino acid sequence having substitution, deletion, insertion, and/or addition of one to ten amino acid residues in the amino acid sequence SEQ ID NO:2, wherein said protein has a D-aminotransferase activity. 
 
     
     
       2. An isolated DNA encoding a protein having an amino acid sequence selected from the following (A) and (B):
 (A) an amino acid sequence having substitution of an amino acid residue at a position selected from positions 100, 180 to 183, 243 and 244, or combinations thereof, in the amino acid sequence of SEQ ID NO:2; and 
 (B) an amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues at position(s) other than positions 100, 180 to 183, 243 and 244 in the amino acid sequence (A), such that a total of one to ten amino acid residues of the amino acid sequence of SEQ ID NO:2 have been altered; 
 wherein said protein has a D-aminotransferase activity, and wherein an amount of (2R, 4R)-monatin produced with said protein from 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid is greater than that produced with a protein having the amino acid sequence of SEQ ID NO:2. 
 
     
     
       3. A recombinant DNA obtained by ligating the DNA according to  claim 2  to a vector DNA. 
     
     
       4. An isolated cell transformed with the recombinant DNA according to  claim 3 . 
     
     
       5. A method for producing a protein having a D-aminotransferase activity, comprising culturing the cell according to  claim 4  in a medium and accumulating said protein having said D-aminotransferase activity in said medium and/or said cell. 
     
     
       6. The isolated DNA according to  claim 1 , wherein said DNA has the nucleotide sequence of SEQ ID NO:1. 
     
     
       7. A recombinant DNA obtained by ligating the DNA according to  claim 6  to a vector DNA. 
     
     
       8. An isolated cell transformed with the recombinant DNA according to  claim 7 . 
     
     
       9. A method for producing a protein having a D-aminotransferase activity, comprising culturing the cell according to  claim 8  in a medium and accumulating said protein having said D-aminotransferase activity in said medium and/or said cell. 
     
     
       10. The isolated DNA according to  claim 1 , wherein said DNA encodes a protein having D-aminotransferase and has a nucleotide sequence which hybridizes under stringent conditions with another DNA composed of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:1, wherein said stringent conditions comprise 65° C., 0.1×SSC and 0.1% SDS. 
     
     
       11. A recombinant DNA obtained by ligating the DNA according to  claim 10  to a vector DNA. 
     
     
       12. An isolated cell transformed with the recombinant DNA according to  claim 11 . 
     
     
       13. A method for producing a protein having a D-aminotransferase activity, comprising culturing the cell according to  claim 12  in a medium and accumulating said protein having said D-aminotransferase activity in said medium and/or said cell. 
     
     
       14. The isolated DNA according to  claim 1 , wherein said DNA has a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2. 
     
     
       15. A recombinant DNA obtained by ligating the DNA according to  claim 14  to a vector DNA. 
     
     
       16. An isolated cell transformed with the recombinant DNA according to  claim 15 . 
     
     
       17. A method for producing a protein having a D-aminotransferase activity, comprising culturing the cell according to  claim 16  in a medium and accumulating said protein having said D-aminotransferase activity in said medium and/or said cell. 
     
     
       18. The isolated DNA according to  claim 1 , wherein said DNA has a nucleotide sequence encoding a protein having an amino acid sequence having substitution, deletion, insertion, and/or addition of one to ten amino acid residues in the amino acid sequence of SEQ ID NO:2, wherein said protein has a D-aminotransferase activity. 
     
     
       19. A recombinant DNA obtained by ligating the DNA according to  claim 18  to a vector DNA. 
     
     
       20. A cell transformed with the recombinant DNA according to  claim 19 . 
     
     
       21. A method for producing a protein having a D-aminotransferase activity, comprising culturing the cell according to  claim 20  in a medium and accumulating said protein having said D-aminotransferase activity in said medium and/or said cell. 
     
     
       22. A method for producing an optically active glutamic acid derivative, comprising:
 (a) expressing a recombinant DNA comprising the DNA of  claim 1  in a cell containing said recombinant DNA to produce said protein having a D-aminotransferase activity; 
 (b) reacting a keto acid represented by the following general formula (1): 
 
       
         
           
           
               
               
           
         
         in the presence of said protein and an amino donor, to generate a (2R, 4R) isomer of said glutamic acid derivative represented by the following general formula (2): 
       
       
         
           
           
               
               
           
         
       
       or a salt thereof;
 wherein R in the formulae (1) and (2) is an aromatic or heterocyclic ring, and said aromatic or heterocyclic ring may further have one or more of a halogen atom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxy group having up to 3 carbon atoms, and an amino group. 
 
     
     
       23. The method for producing the optically active glutamic acid derivative according to  claim 22 , wherein said R is a phenyl or indolyl group. 
     
     
       24. The method for producing the optically active glutamic acid derivative according to  claim 22 , wherein said amino donor is an amino acid. 
     
     
       25. The method for producing the optically active glutamic acid derivative according to  claim 24 , wherein said reaction is performed in a reaction system further containing an enzyme having an activity to catalyze a reaction for converting an L-amino acid to a D-amino acid, or a microorganism having such an enzymatic activity. 
     
     
       26. The method for producing the optically active glutamic acid derivative according to  claim 22 , further comprising purifying said protein prior to said reacting. 
     
     
       27. The method for producing the optically active glutamic acid derivative according to  claim 22 , further comprising isolating said optically active glutamic acid derivative. 
     
     
       28. The isolated DNA according to  claim 2 , wherein said DNA has a nucleotide sequence encoding a protein having an amino acid sequence selected from (A) or (B):
 (A) an amino acid sequence having substitution selected from the group consisting of the following substitutions (a) to (e), or a combination thereof, in an amino acid sequence of SEQ ID NO: 2: 
 (a) substitution of a serine residue at position  181  with another amino acid residue; 
 (b) substitution of an alanine residue at position  182  with another amino acid residue; 
 (c) substitution of an asparagine residue at position  183  with another amino acid residue; 
 (d) substitution of a serine residue at position  243  with another amino acid residue; and 
 (e) substitution of a serine residue at position  244  with another amino acid residue; and 
 (B) an amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues at position(s) other than positions  181  to  183 ,  243 , and  244  in the amino acid sequence (A), such that a total of one to ten amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 have been altered. 
 
     
     
       29. The isolated DNA according to  claim 28 , wherein said group consisting of said substitutions (a) to (e) is the group consisting of the following substitutions (a) to (e):
 (a) substitution of the serine residue at position  181  with an aspartic acid residue; 
 (b) substitution of the alanine residue at position  182  with a lysine or serine residue; 
 (c) substitution of the asparagine residue at position  183  with a serine residue; 
 (d) substitution of the serine residue at position  243  with a glutamic acid, leucine, lysine, asparagine or glutamine residue; and 
 (e) substitution of a serine residue at position  244  with a lysine residue. 
 
     
     
       30. The isolated DNA according to  claim 2 , wherein said DNA has a nucleotide sequence encoding a protein having an amino acid sequence selected from (A) or (B):
 (A) an amino acid sequence having substitution selected from the group consisting of the following substitutions (a) to (e), or a combination thereof, in the amino acid sequence of SEQ ID NO: 2: 
 (a) substitution of an asparagine residue at position  100  with another amino acid residue; 
 (b) substitution of a serine residue at position  181  with another amino acid residue; and 
 (c) substitution of an alanine residue at position  182  with another amino acid residue; and 
 (B) an amino acid sequence having substitution, deletion, insertion, and/or addition of one or several amino acid residues at position(s) other than positions  100 ,  181 , and  182  in the amino acid sequence (A), such that a total of one to ten amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 have been altered. 
 
     
     
       31. The isolated DNA according to  claim 30 , wherein said group consisting of said substitutions (a) to (c) is the group consisting of the following substitutions (a) to (c):
 (a) substitution of the asparagine residue at position  100  with an alanine residue; 
 (b) substitution of the serine residue at position  181  with an alanine residue; and 
 (c) substitution of the alanine residue at position  182  with a serine residue. 
 
     
     
       32. The isolated DNA according to  claim 2 , wherein said DNA has a nucleotide sequence encoding a protein comprising an amino acid sequence having substitution selected from the group consisting of the following substitutions (i) to (vii), or a combination thereof, in the amino acid sequence of SEQ ID NO: 2:
 (i) substitution of a serine residue at position  243  with an asparagine residue; 
 (ii) substitution of a serine residue at position  244  with a lysine residue; 
 (iii) substitution of a serine residue at position  180  with an alanine residue and substitution of a serine residue at position  243  with an asparagine residue; 
 (iv) substitution of a serine residue at position  180  with an alanine residue and substitution of a serine residue at position  244  with a lysine residue; 
 (v) substitution of a serine residue at position  243  with an asparagine residue and substitution of a serine residue at position  244  with a lysine residue; 
 (vi) substitution of an asparagine residue at position  100  with an alanine residue and substitution of a serine residue at position  243  with an asparagine residue; and 
 (vii) substitution of an alanine residue at position  182  with a serine residue and substitution of a serine residue at position  243  with an asparagine residue. 
 
     
     
       33. A method for producing an optically active glutamic acid derivative, comprising:
 (a) expressing a recombinant DNA comprising the DNA of  claim 2  in a cell containing said recombinant DNA to produce said protein having a D-aminotransferase activity; 
 (b) reacting a keto acid represented by the following formula (1): 
 
       
         
           
           
               
               
           
         
         in the presence of the protein and an amino donor, to generate a (2R, 4R) isomer of said glutamic acid derivative represented by the following formula (2): 
       
       
         
           
           
               
               
           
         
       
       or a salt thereof;
 wherein R in the formulae (1) and (2) is an aromatic or heterocyclic ring, and said aromatic or heterocyclic ring may further have one or more of a halogen atom, a hydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxy group having up to 3 carbon atoms, and an amino group. 
 
     
     
       34. The method according to  claim 33 , wherein said R is a phenyl or indolyl group. 
     
     
       35. The method according to  claim 33 , wherein said amino donor is an amino acid. 
     
     
       36. The method according to  claim 35 , wherein said reaction is performed in a reaction system further containing an enzyme having an activity to catalyze a reaction for converting an L-amino acid to a D-amino acid, or a microorganism having such an enzymatic activity. 
     
     
       37. The method according to  claim 33 , further comprising purifying said protein prior to said reacting. 
     
     
       38. The method according to  claim 33 , further comprising isolating said optically active glutamic acid derivative.

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